Induction of tolerance by apoptotic and/or necrotic cells

ABSTRACT

The present invention is directed to a method of inducing tolerance to self-antigens in a subject having an autoimmune diseases. In particular, the invention provides a pharmaceutical composition and method of use thereof for the modulation of immunogical activity in an animal subject. Said modulation may be an increased tolerance to self apoptotic cells, a reduction in the tissue levels of autoantibodies associated with apoptotic cells, a reduction in the tissue levels of autoantibodies associated with an autoimmune disease, a reduction in the level of inflammation and inflammatory mediators associated with an autoimmune disease, a reduction in the level of tissue damage associated with an autoimmune disease, or a combination thereof.

FIELD OF THE INVENTION

This invention relates to the field of medicine, in particular to thetreatment of diseases which arise due to malfunctioning of the immunesystem, such as autoimmune diseases. The invention relates as well topharmaceutical compositions comprising apoptotic and/or necrotic cellswhich are useful for treating such diseases.

BACKGROUND OF THE INVENTION

The immune system of animals is a complex and multivarious networkcomprising cells, antibodies, solid and non-solid organs, and chemicalmessenger molecules which allow for communication between thesestructures. A hallmark of a healthy immune system is the ability torecognize bacteria, viruses, and other foreign bodies and to effectivelyattack such pathogens while continuing to distinguish between theforeign bodies and the molecules, cells, tissues and organs comprisingthe individual organism. When this aspect of an animal's immune systemis deficient the result is a state of disease, often one in which theimmune system attacks one or more specific molecules or cells leading totissue and organ damage. Since the immune system destroys bodiesrecognized as foreign, often known as antigens, through a complexprocess known as inflammation, of which many different types exist, theimmediate and chronic types of tissue damage in autoimmune andinflammatory diseases are frequently the result of one or more types ofinflammation.

In autoimmune disease an immune response directed against one or morecomponents of the animal's own tissues or cells results in damage to oneor more organs or tissues. In mammals, particularly in humans, manyclinically different types of autoimmune disease occur, includingsubtypes of particular autoimmune disease. Although each type ofautoimmune disease is associated with a spectrum of clinical symptomsand aberrant laboratory parameteres, signs and symptoms of autoimmunediseases frequently overlap so that one or more are diagnosed in thesame patient. The vast majority cases in which one or more autoimmunedisease has been diagnosed are characterized by the presence in theaffected subject of autoantibodies. The autoantibodies are directed toone or more molecular or cellular targets, known as antigens, within theanimal. Such autoantibodies are present at tissue levels, which areoften ten to one hundred times the normal level in healthy individualsand give rise to a significant proportion of the organ and tissue damageassociated with the particular autoimmune disease. For example, in theautoimmune disease myasthenia gravis, autoantibodies against a receptorin meuromuscular junction are associated with muscle weakness, while insystemic lupus erythematosus, anti-dsDNA antibodies are associated withnephritis in human patients and can cause nephritis upon injection tonormal mice. The tissue and organ damage is attributed to the presenceof autoantibodies and to the inflammation, which arises to dueinflammatory immune responses, set off by autoantibodies. Thus the signsand symptoms of disease are due to autoantibodies, the autoimmuneinflammatory response, or a combination thereof.

Autoimmune diseases include rheumatoid arthritis, graft versus hostdisease, systemic lupus erythromatosus (SLE), scleroderma, multiplesclerosis, diabetes, organ rejection, inflammatory bowel disease,psoriasis, and other afflictions. It is becoming increasingly apparentthat many vascular disorders, including atherosclerotic forms of suchdisorders, have an autoimmune component, and a number of patients withvascular disease have circulating autoantibodies. Autoimmune diseasesmay be divided into two general types, namely systemic autoimmunediseases (exemplified by lupus and scleroderma), and organ specific(exemplified by multiple sclerosis, diabetes and atherosclerosis, inwhich latter case the vasculature is regarded as a specific organ).

One important autoimmune disease is lupus and this disease is a modelfor enraveling the physiology and developing inventive treatments forautoimmune disease in general. It has long been appreciated that DNA andhistones are major autoantigens in systemic lupus erythematosis (SLE).However, only recently has evidence been provided that the DNA-histonecomplex, i.e., nucleosomes, are the preferred targets of autoantibodiesin SLE. The question then arises as to how nucleosomes and several otherintracellular antigen targets can be immunogenic in SLE. Duringapoptosis, the membrane of cells undergoing apoptosis form cytoplasmicblebs, some of which are shed as apoptotic bodies. It was recentlydemonstrated that exposure of kertinocytes to high frequency lightinduces apoptosis, and that the cell surface expression of Ro and La,but also of nucleosomes and ribosomes, can be explained by translocationof certain intracellular particles to the apoptotic surface blebs.Significantly, another translocation which occurs during apoptosis isthat of phosphatidyl serine (PS), an acidic phospholipid that normallyresides on the inside of the cell, but flips to the outside of the cellmembrane when the cell undergoes apoptosis. PS like, cardiolipin, is amajor autoantigen for anti-phospholipid (aPL) antibodes in SLE. Takentogether, these findings provide a unifying hypothesis to explainantigen selection in SLE, e.g., that SLE patients are responding to theexposure of intracellular proteins translocated to the cell surfaceduring apoptosis.

Thus, SLE patients form an immune response to apoptotic material.Although there may be many possible explanations to explain thisobservation, any explanation must take into account that in SLE patientsthe uptake of apoptotic cells by macrophages in vitro is reduced.Furthermore, brief, limited administration of syngeneic apoptotic cellsto normal strains of mice leads to induction of autoantibodies andglomerular depositions. In addition, it has been shown that thecomplement system is important in clearance of uptake of apoptoticcells, suggesting the novel hypothesis disclosed herein for the reasonwhy greater than 90% of patients homozygous for C1q and greater than 70%of C4 deficiency patients develop SLE.

This novel understanding of the pathogenesis of SLE may suggest adifferent approach to the treatment of SLE. Manipulation of the immunesystem to prevent a deleterious response has been the goal ofimmunologists for many years in transplantation biology and autoimmunediseases. Traditionally, the main effort was to induce immunosuppressionand the current therapy for a classical systemic autoimmune disease suchas SLE is drug treatment with corticosteroids, azathyoprine,cyclopohosphamide, and cyclosporine, all of which are administered withthe aim suppressing the immune system. Immunosuppression was animportant step in ameliorating the 5-years survival rate of SLE patientsin the last three decades but it is far from the ideal treatment sinceno cure is achieved and patients suffer from very serious side effectsleading to high rates of morbidity and being the main cause of prematuremortality. In that regard, even the newly developed biologics currentlyunder toxicity and efficacy evaluation, such as anti-CD40 ligand, andCTLA4 lg, are non-specific for the autoimmune B and T cell clones and,if successful for autoimmunity, will suppress probably the entire immunesystem.

In addition to fighting infections, the immune system has other roles inmaintaining the normal state of health and function of the animal.Throughout the life span of an animal, tissues become reshaped withareas of cells being removed. This is accomplished by the cells'undergoing a process called programmed cell death or apoptosis, theapoptotic cells disintegrating and being phagocytosed while not becomingdisrupted. In many organs, for example, a certain percentage of thecells die off every day while different branches of the immune systemare typically called in to remove the dead cells and parts thereof tomake room for the new cells which are born to replace them. Were it notfor the cellular debri removing cells of the immune system, often knownas macrophages, tissue and organ growth would be impossible due to alack for space.

In fact, the process of apoptosis is considered to be particularlyimportant in the development and maintenance of the immune systemitself, where the immune cells which recognize or attack other normalcells of the animal are destroyed and removed by this process. Thus,while apoptosis is a process used by the immune system in protecting theorganism, it is also used to maintain tolerance to self antigens andtherefore allowing the immune system to fulfill its role indistinguishing the animal's own cells from those of non-self invaders.

Immature dendritic cells (IDC) engulf apoptotic cells and are able toacquire antigens found in the dying cells. IDC that capture apoptoticmacrophages infected by killed influenza-virus, mature and activatelymphocytes to mount virus-specific CTL responses in the presence ofconditioned media. However, in the absence of infection and conditionedmedia, IDC do not mature following uptake of apoptotic cells and as aconsequence are less able to efficiently present acquired antigens.Furthermore, it has been suggested that following interaction withapoptotic material, IDC may have a role in maintaining peripheraltolerance to self-antigens that are permanently created at differentsites. In support of this, autoimmunity or lupus like disease has beenobserved in mice and human deficient in receptors important for uptakeof apoptotic cells such as ABC1 cassette transporter, Mer, andcomplement deficiencies. Clearance via specific receptors may dictatespecific immune response or tolerance as demonstrated by TGF-β and IL-10secretion by macrophages following uptake by macrophages. So, cytokines,chemokines, eicosanoids, and additional materials found in the milieu ofthe interaction, may polarize the immune response.

Thus, the aim of the present Invention is to induce tolerance to selfantigens in a subject having an autoimmune disease, mainly antigensrelated to apoptotic and/or necrotic cells.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 demonstrates the modulation of Immune Response inMRL/MpJ-Fas^(Ipr) Mice Following Injection With Apoptotic Cells vsPlacebo—Decrease in anti-single stranded DNA Antibodies

FIG. 2 demonstrates the modulation of Immune Response inMRL/MpJ-Fas^(Ipr) Mice Following Injection With Apoptotic Cells vsPlacebo—Decrease in anti-double stranded DNA Antibodies

SUMMARY OF THE INVENTION

The present invention is directed to a method of inducing tolerance toself-antigens in a subject having an autoimmune disease. According tothe present invention, a lack of tolerance to apoptotic and/or necroticcells is an important aspect in the development of autoimmune diseaseand an important target for therapy.

In particular, the invention provides a pharmaceutical composition andmethod of use thereof for the modulation of immunogical activity in ananimal subject wherein said modulation is an increased tolerance toapoptotic and/or necrotic cells, a reduction in the tissue levels ofautoantibodies associated with apoptotic and/or necrotic cells, areduction in the tissue levels of autoantibodies associated with anautoimmune disease, a reduction in the level of inflammation andinflammatory mediators associated with an autoimmune disease, areduction in the level of tissue damage associated with an autoimmunedisease, or a combination thereof.

A composition for treating autoimmune diseases according to the presentinvention should contain antigens, i.e. apoptotic and/or necrotic cells,or fragments thereof, i.e. blebs of apoptotc cells, membrane fragments,and peptides, that upon administration, interact with the immune systemof the animal to produce enhanced tolerance to self antigens. Inaddition, the antigens or fragment thereof should be present in a formwhich can be recognized by the subject's immune system when thecomposition is administered to the subject. The desirable antigens maybe present on intact apoptotic cells or necrotic cells on fragmentsthereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition and methodof use thereof for the modulation of immunogical activity in an animalsubject wherein said modulation is an increased tolerance to apoptoticand/or necrotic cells. A composition for treating autoimmune diseasesaccording to the present invention should contain antigens, i.e.apoptotic and/or necrotic cells, or fragments thereof, i.e. blebs ofapoptotic cells, membrane fragments, and peptides, that uponadministration, interact with the immune system of the animal to produceenhanced tolerance to self antigens.

Antibody isotype switching is a process whereby an immune cell producingone type of antibody isotype directed against a particular antigenbegins to produce a second antibody isotype directed against the sameantigen. One example of antibody switching occurs in the transition fromacute to chronic infection, when a lymphocyte producing an IgG isotypedirected an antigen of an infectious invading agent begins to produce anIgM isotype directed against the same antigen, which often signifies theacquisition of an immune state toward the infectious agent. Antibodyisotype switching is known to occur in other situations including, butnot only, autoimmune states wherein isotype switching occurs for theproduction of antibodies, i.e. autoantibodies, directed againstself-antigens.

According to the present invention, an autoimmune disease is, but notonly, one of the following: systemic lupus erythematosis, discoid lupuserythematosis, rheumatoid arthritis, diabetes mellitus, graft versushost disease, scleroderma, multiple sclerosis, diabetes, organrejection, inflammatory bowel disease, psoriasis, miscarriage,infertility, atherosclerosis, and other inflammatory disorders.

Examples of systemic autoimmune disease are rheumatoid arthritis, lupusand scleroderma, and examples of organ specific autoimmune disease aremultiple sclerosis, diabetes and atherosclerosis.

According to the present invention, tolerance is the ability of theimmune system to properly recognize self-antigens in a way which doesnot produce signs or symptoms of an autoimmune disease. The enhancedtolerance to apoptotic and/or necrotic antigens may be obtained by areduction in the presentation of apoptotic and/or necrotic antigens ondendritic cells.

According to the present invention, administering to a subject acomposition comprising apoptotic and/or necrotic cells as antigen is amean of inducing tolerance and thereby reducing the signs or symptoms ofautoimmune disease, in particular a disease associated with adisturbance in the process of apoptosis or clearance of apoptotic and/ornecrotic cells.

According to the present invention, a composition to treat autoimmunediseases contains antigens or fragments thereof (peptides) that willenhance the immune mechanism of tolerance to the self-antigens which areassociated with the autoimmune disease. Such antigens may be present onapoptotic and/or necrotic cells or released by apoptotic and/or necroticcells, or a combination thereof. Administering a composition comprisingapoptotic and/or necrotic cells is a mean of making available to theimmune system of a subject an additional amount of said antigens so asto provide for the development of tolerance to said antigens in thesubject. An alternative or parallel explanation for the efficacy of thecomposition of the present invention in treating an autoimmune diseasewhen administered to a subject afflicted thereby, is that administrationof apoptotic and/or necrotic cells invokes an beneficial immune responsein addition to that of tolerance induction or enhanced apoptotic cellclearance.

According to the present invention, an autoimmune response is an immuneresponse directed against one or more self-antigens of an animal.

According to the present invention, apoptosis is a process of programmedcell death, wherein the cell enters a stage characterized by thebreakdown or disappearance of cellular components essential tomaintainance of the normal differentiated state of the cell, whilemaintaining an intact, non-porous membrane. In one aspect of apoptosis,the cell undergoes a process of de-differentiation whereby the abilityto remain in a functional, viable, differentiated state is lost. Thepathway of apoptosis may include, but not only, loss of membranepotential of the mitochondria, activation of serine proteases,activation of caspases, activation of other proenzymes, cleavage ofproteins, cleavage of DNA, cleavage of DNA to form nucleosomes,phosphorylation of proteins, exposure of phosphatidylserine residues onthe outer membrane surface, the appearance of blebs, condensedchromatin, contracted cytoplasm, or a combination thereof. Of theforegoing, early signs of apoptosis include, but not only, loss ofmembrane potential of the mitochondria, cleavage of proteins, cleavageof DNA, and protein phosphorylation and exposure of phosphatidylserine.Nectosis is the feature of cells undergoing death that leads among otherthings to disruption of the membrane and swelling of the cell.

In one embodiment, the present invention provides a method of treating asubject having an autoimmune disease, comprising the steps of: obtainingcells from the subject; inducing cell death in said cells resulting inapoptotic and/or necrotic cells; administering to the subject an amountof said apoptotic and/or necrotic cells effective to produce a modifiedimmune response in said subject, thereby treating the subject with theautoimmune disease.

In another embodiment, the present invention provides a method oftreating a subject having an autoimmune disease, comprising the step ofadministering to the subject an amount of apoptotic and/or necroticcells effective to produce a modified immune response in said subject,thereby treating the subject with the autoimmune disease.

According to the present invention, an apoptosis-inducing agent is anatural or synthetic molecule, a natural or synthetic antibody, animmunological cell, gamma- or UV-irradiation, in the presence of which,or upon contact by which, a cell becomes an apoptotic cell. Examples ofapoptosis-inducing agents are immunosuppressive drugs including, but notonly, corticosteroids, cyclophosphamide, methotrexate, azothioprine,cyclosporine, staurosporine, or a combination thereof. Necrosis can beinduced by H₂O₂ or heating or using other methods known in the art.

According to the present invention, an apoptosis and/or necrosisinducing treatment is a set of one or more environmental conditions inwhich a cell becomes an apoptotic and/or necrotic cell. Examples ofapoptosis-inducing treatments are U.V.- or gamma-irradiation, heating,cooling, serum deprivation, growth factor deprivation, acidifying,diluting, alkalizing, ionic strength change, serum deprivation,irradiating, or a combination thereof.

According to the present invention, induction of apoptosis occurs when acell becomes an apoptotic cell. For induction of apoptosis in anyparticular cell or cells, the choice of apoptosis-inducing agent andapoptosis-inducing treatment to yield an apoptotic cell or cells is afunction of the type of cell, tissue of origin, and means of obtainingthe cell sample. Apoptosis may be induced in vivo, in situ, in vitro, orex vivo. Some of the commonly known apoptosis-inducing methods arecontacting a cell with a steroid, such as dexamethasone, exposing a cellto radiation, in particular gamma-radiation, exposing a cell toconditions of serum deprivation, in particular 0-5% serum, contacting acell with perforin, or a combination thereof. In addition to inducingapoptosis, many of these agents and conditions produce as well, invarying proportions, induction of necrosis, otherwise known asaccidental cell death, necrotic cells (primary or secondary), andotherwise damaged, non-apoptotic cells. Thus, for purposes of thepresent invention, apoptotic cells may comprise at any one time bothapoptotic cells and fragments thereof, and as well a certain percentageof necrotic or lysed cells and fragments thereof.

The presence of an apoptotic cell may be confirmed by DNAelectrophoresis, TUNEL (DNA labeling), annexin-FITC plus propidiumiodide, propidium iodide staining of fragmented DNA (hypodipoid region),caspase activation, cleavage of target proteins, morphologically usinglight microscopy with appropriate staining, electron microscopy, or acombination thereof.

According to the present invention, an apoptotic cell is a cell in whichapoptosis has been induced either through the course of a diseaseprocess or induced through contact with an apoptosis-inducing agent,exposure to an apoptosis-inducing treatment, or a combination thereof.

According to the present invention, apoptotic and/or necrotic cells arepreferably obtained through a method comprising the steps of: obtainingcells concentrated from blood; contacting the cells with anapoptosis-inducing agent or with a necrosis-inducing agent andincubating at 37° C. in a physiologically suitable medium.

According to the present invention, an apoptosis-inducing agent is anatural or synthetic molecule, a natural or synthetic antibody, animmunological cell, gamma- or UV-irradiation, in the presence of which,or upon contact by which, a cell becomes an apoptotic cell. Examples ofapoptosis-inducing agents are immunosuppressive drugs including, but notonly, corticosteroids, cyclophosphamide, methotrexate, azothioprine,cyclosporine, staurosporine, or other compounds or a combinationthereof. Necrosis can be induced by H₂O₂ or heating or with othermethods known in the art.

According to the present invention, an apoptosis- and/ornecrosis-inducing treatment is a set of one or more environmentalconditions in which a cell becomes an apoptotic and/or necrotic cell.Examples of apoptosis-inducing treatments are U.V.- orgamma-irradiation, heating, cooling, serum deprivation, growth factordeprivation, acidifying, diluting, alkalizing, ionic strength change,serum deprivation, irradiating, or a combination thereof.

According to the present invention, induction of apoptosis occurs when acell becomes an apoptotic cell. For induction of apoptosis in anyparticular cell or cells, the choice of apoptosis-inducing agent andapoptosis-inducing treatment to yield an apoptotic cell or cells is afunction of the type of cell, tissue of origin, and means of obtainingthe cell sample. Apoptosis may be induced in vivo, in situ, in vitro, orex vivo. Some of the commonly known apoptosis-inducing methods arecontacting a cell with a steroid, such as dexamethasone, exposing a cellto radiation, in particular gamma-radiation, exposing a cell toconditions of serum deprivation, in particular 0-5% serum, contacting acell with perforin, or a combination thereof. In addition to inducingapoptosis, many of these agents and conditions produce as well, invarying proportions, induction of necrosis, otherwise known asaccidental cell death, necrotic cells (primary or secondary), andotherwise damaged, non-apoptotic cells. Thus, for purposes of thepresent invention, apoptotic cells may comprise at any one time bothapoptotic cells and fragments thereof, and as well a certain percentageof necrotic or lysed cells and fragments thereof.

The presence of an apoptotic cell may be confirmed by DNAelectrophoresis, TUNEL (DNA labeling), annexin-FITC plus propidiumiodide, propidium iodide staining of fragmented DNA (hypodipoid region),caspase activation, cleavage of target proteins, morphologically usinglight microscopy with appropriate staining, electron microscopy, or acombination thereof.

According to the present invention, an apoptotic cell is a cell in whichapoptosis has been induced either through the course of a diseaseprocess or induced through contact with an apoptosis-inducing agent,exposure to an apoptosis-inducing treatment, or a combination thereof.

In one aspect of the composition and methods of the invention, the cellsare derived from the subject to be treated (autologous source), so as toavoid the possibility of contamination with undesirable infectiousagents which may be present in donors. Thus, in one embodiment, thecomposition comprises apoptotic cells which are derived from cellsobtained from the subject to be treated. For obtaining cells, virtuallyany technique for obtaining cells may be used, with the exception ofprocedures which markedly interfere with the potential for a cell tobecome an apoptotic cell or which interfere with the potential of anapoptotic cell to induce tolerance. Furthermore, the use of anautologous source of cells is preferable since apoptotic cells obtainedtherefrom are most likely to invoke the desired response of toleranceand are most likely to contain the desired antigen configuration.

Thus, a therapeutic composition comprising apoptotic and/or necroticcells which are able to exert a strong influence on the immune systemand encourage the development of tolerance. For purposes of the presentinvention, the apoptotic cells may be rendered more highly activethrough modifications which enhance immunogenecity. Thus antigenicapoptotic cells may have a more avid interaction with the immune systemwhen administered with an immunosuppressing molecules such as IL-10 orother immunosuppressing cytokines, chemokines or other peptides ormolecules. Other procedures which are known to enhance immunogenecityare linking together, i.e. cross-linking, or linking with other ligandsdirectly or through spacers.

According to the present invention, an apoptotic and/or necrotic cellmay derive from any body tissue, soft tissue, solid tissue, lymphatictissue or hematogenous tissue. The cell may be of syngeneic origin orpedigree, autologous origin or pedigree, allogenic origin or pedigree,xenogenic origin or pedigree or a combination thereof. An apoptoticand/or necrotic cell may be derived from a cell obtained from a bodytissue, including but not only, the following: blood, sputum, lymph,lymph node, thymus, bone marrow, saliva, dermis, epidermis, hypodermis,mucosa, submucosa, an internal organ, connective tissue, muscle, smoothmuscle, synovial fluid, spinal fluid, or a combination thereof.

According to the present invention, for the purpose of obtaining cells,a cell or cells may be obtained from a body tissue by a tissue biopsy;an exfoliative biopsy; a fine needle biopsy; a blood extract; a bonemarrow tap; a lymph node biopsy; a lymph node aspirate; a thymus biopsy;a thymus aspirate; a synovial fluid aspiration; a bronchial lavage; aperitoneal lavage; a peritoneal tap; a pleural tap; a spinal fluid tap;a body tissue stored ex vivo; a tissue culture; a skin biopsy; ascraping; an exfoliation; a mucosal biopsy; a mucosal scraping; a cellculture; a cultured cell line; a cultured cell line comprising apoptoticcells; a cultured cell line comprising a gene library; transformedcells; transgenic cells; a cell modified through insertion of atransgene; or a combination thereof. Cells may be of syngeneic origin orpedigree, autologous origin or pedigree, allogenic origin or pedigree,xenogenic origin or pedigree, or a combination thereof.

According to the present invention, a transformed cell is a cell, or anancestor thereof, into which has been introduced, by means ofrecombinant DNA techniques, a DNA molecule encoding a desired gene.

According to the present invention, a transgene is any piece of DNAwhich is inserted by artifice into a cell, and becomes part of thegenome of the organism which develops from that cell. Such a transgenemay include a gene which is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism.

According to the present invention, a transgenic cell is a cell whichincludes a DNA sequence which is inserted by artifice into the cell andbecomes part of the genome of the organism which develops from thatcell. As used herein, the transgenic organisms are generally transgenicmammalian (e.g., rodents such as rats or mice) and the DNA (transgene)is inserted by artifice into the nuclear genome.

According to the present invention, elements of the immune system of ananimal include, but not only, the following: antibodies, chemokines,eukocytes, lymphocytes, T-cells, B-cells, plasma cells, granulocytes,neutrophils, macrophages, monocytes, eosinophils, platelets, dendriticcells, antigen presenting cells, or a combination thereof.

According to the present invention, a modified immune response is achange in the amount, level, rate of synthesis, rate of degradation,pattern of distribution, systemic concentration, localized concentrationof one or more elements of the immune system of the organism in one ormore tissues of the organism.

According to the present invention, modulation of an immune response isan action which results in a modified immune response.

According to the present invention, modulation of an autoimmune responseis an action which results in a modified autoimmune response.

According to the present invention, treatment of a disease is an actionwhich results in a reduction in the severity of one or more signs orsymptoms of said disease including, but not only, a reduction in thelevels of autoantibodies, a reduction in the levels of inflammatorymediators, a reduction in inflammation, a reduction in tissue damage,subjective relief from any symptom attributed to the disease includingsubjective relief from pain or discomfort reported by the subject, or acombination thereof.

Method of Treatment

In one embodiment, the present invention provides a method of treating asubject having an autoimmune disease, comprising the steps of: obtainingcells from the subject; inducing cell death in said cells resulting inapoptotic and/or necrotic cells; administering to the subject an amountof said apoptotic and/or necrotic cells effective to produce a modifiedimmune response in said subject, thereby treating the subject with theautoimmune disease.

In another embodiment, the present invention provides a method oftreating a subject having an autoimmune disease, comprising the step ofadministering to the subject an amount of apoptotic and/or necroticcells effective to produce a modified immune response in said subject,thereby treating the subject with the autoimmune disease.

In one embodiment, induction of cell death is obtained by exposing saidcells to an apoptosis-inducing agent, apoptosis-inducing treatment, anecrosis-inducing agent or a necrosis-inducing treatment

In one embodiment, the modified immune response is an increasedtolerance to self-apoptotic cells or a reduction in the tissue level ofauto-antibodies associated with self-apoptotic cells. Saidauto-antibodies may be antinuclear antibodies, anti-single stranded DNAantibodies, anti-double stranded DNA antibodies, anti-cardiolipinantibodies, anti-phosphatidylserine antibodies, anti-2GPI antibodies,anti-Sm antibodies, anti-RNP antibodies, or anti-Ku antibodies.

In another embodiment, the modified immune response in said subject is areduction in the level of inflammatory response. Said inflammatoryresponse may be associated with chemokines, cytokines, eicosanoids,complement proteins, C-reactive protein, TNF, or a combination thereof.

In another embodiment, the autoimmune disease is associated with animmune response to self-antigens appearing on apoptotic cells. Saidautoimmune disease may be systemic or discoid lupus, erythematosis,rheumatoid arthritis, polymyositis, or vasculitis.

In one embodiment, the cells of the invention are derived fromautologous origin. Said cells may be derived from hematopoetic cells,thymocytes, splenocytes, lymphocytes, monocytes, or a combinationthereof. In another embodiment, the cells of the invention may bederived from any other sources such as cell lines.

In another embodiment, the amount of said composition comprisingapoptotic cells is at least one hundred apoptotic cells of apharmaceutically acceptable fragment thereof, per kg body weight, incombination with a pharmaceutically acceptable carrier.

In one embodiment, the apoptosis-inducing agent is a steroid, a peptide,a protein, a sugar, a lipid, an antibody, or a combination thereof. Saidsteroid may be for example dexamethasone. Said protein may be forexample perforin.

In another embodiment, the apoptosis-inducing treatment is cooling,heating, acidifying, diluting, alkalizing, ionic strength changing,serum deprivating, irradiating, or a combination thereof.

In one embodiment of the method of the invention, the compositioncomprising apoptotic cells is administered intravenously, intradermally,subdermally, intramuscularly, orally or a combination thereof. Thecomposition may be administered in combination with an immunosuppressingmolecules such as IL-10 or TGF-β.

In a preferred embodiment of the method of the invention, the cells areobtained from the blood of the subject to be treated through separationof said blood into fractions, resuspension of the fraction containingthe desired cells in a physiologically acceptable buffered medium,adding an apoptosis-inducing agent under conditions which induceapoptosis, such as an apoptosis-inducing treatment while maintaining thepH, ionic strength, and temperature at physiologically acceptable limitsto form a composition comprising apoptotic cells; the composition isinjected into the subject via an effective intra- or extravascular routein an amount of between 500,00 to 50×10⁹ cells per 70 kg human subjectat a frequency of once or twice per day until the desired modifiedimmune response is obtained, thereby treating the subject.

Pharmaceutical Composition

The invention provides a pharmaceutical composition comprising aneffective amount of apoptotic and/or necrotic cells, whereinadministration of said composition to a subject produces a modifiedimmune response in said subject.

According to the present invention, a pharmaceutical compositioncomprising apoptotic cells may be obtained by subjecting said cells toan apoptosis-inducing agent, an apoptosis-inducing treatment, or acombination thereof.

In one embodiment, a composition comprising apoptotic and/or necroticcells is produced by contacting said cells with an apoptosis-inducingagent or necrosis-inducing agent. In another embodiment, a compositioncomprising apoptotic and/or necrotic cells is produced by exposing saidcells to an apoptosis-inducing treatment or necrosis-inducing treatment.In another embodiment, a composition comprising apoptotic cells isproduced by exposing said cells to an apoptosis-inducing agent before,during, or following exposure of said cells to an apoptosis-inducingtreatment.

In one embodiment, said composition comprises at least one hundredapoptotic and/or necrotic cells. In one embodiment, said compositioncomprises from between one hundred to five billion cells.

In one embodiment of the composition of the invention, said modifiedimmune response is increased tolerance to self-apoptotic cells. Inanother embodiment of the composition of the invention, said modifiedimmune response is a reduction in the tissue levels of autoantibodies insaid subject.

In another embodiment of the composition of the invention, saidautoantibodies are anti-nuclear antibodies, anti-single stranded DNAantibodies, anti-double stranded DNA antibodies, anti-cardiolipinantibodies, anti-phosphatidylserine antibodies, anti-2GPI antibodies,anti-Sm antibodies, anti-RNP antibodies, anti-Ku antibodies, or acombination thereof.

In another embodiment of the composition of the invention, said modifiedimmune response is a reduction in the level of inflammation or tissuedamage, or a combination thereof, in said subject.

In another embodiment of the composition of the invention, saidinflammatory response is associated with chemokines, cytokines,eicosanoids, complement proteins, C-reactive protein, TNF, dendriticcells or a combination thereof.

In another embodiment of the composition of the invention, saidautoimmune disease is associated with an immune response toself-antigens appearing on apoptotic cells.

In another embodiment of the composition of the invention, saidautoimmune disease may be systemic or discoid lupus erythematosis,rheumatoid arthritis, polymyositis, or vasculitis.

In another embodiment of the composition of the invention, the cells arederived from hematopoetic cells, thymocytes, splenocytes, lymphocytes,monocytes, cell lines or a combination thereof.

In another embodiment of the composition of the invention, saidapoptosis-inducing agent is an immunosuppressive medication, including,but not only, the following: azathioprine, cyclophosphamide,methotrexate, prednisone, cyclosporine, or a combination thereof.

In another embodiment of the composition of the invention, saidapoptosis-inducing treatment is cooling, heating, acidifying, diluting,alkalizing, ionic strength change, serum deprivation, irradiating, or acombination thereof.

In another embodiment of the composition of the invention, saidcomposition is suitable for administration via an intravenous route, anintradermal route, a subdermal route, an intramuscular route, or acombination thereof.

In another embodiment of the composition of the invention, saidcomposition is administered in combination with immunosupressingmolecules such as IL-10 or TGF-β.

The composition of the invention may be administered with apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecomposition to patients having an autoimmune disease. Any appropriateroute of administration may be employed, for example, parenteral,intravenous, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, or oraladministration. Therapeutic formulations may be in the form of liquidsolutions or suspensions, prepared fresh or from lyophilized cells.Methods well known in the art for making formulations are found in, forexample, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for composition comprising apoptotic cells includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes.

If desired, treatment with the composition of the invention may becombined with more traditional therapies for the disease such assurgery, radiation, or chemotherapy for cancers; surgery, steroidtherapy, immunosuppresion therapy and chemotherapy for autoimmunediseases or graft vs host disease; antiviral therapies for AIDS; and forexample, tissue plasminogen activator for ischemic injury.

Experimental Results

In the study presented herein, one of the classical models for SLE-likedisease, the MRL/MpJ-Fas^(Ipr), was used for tolerance induction toself-apoptotic cells. These mice develop SLE-like disease due tomutation in Fas, a receptor that mediates apoptosis and activation ofinduced cell death of the immune system. Since in SLE patients, as wellas in MRL/MpJ-Fas^(Ipr) mice, the development of autoantibodies andkidney disease are the most specific pathophysiological parameters,those parameters were evaluated in MRL/MpJ-Fas^(Ipr) following inductionof tolerance to self apoptotic cells.

Methods and Materials

Immunization protocol. MRL/MpJ-Fas^(Ipr) and C3H-SnJ mice were obtainedfrom Jackson Laboratories, Bar Harbor, Me. Thymocytes and splenoscyteswere prepared from 4 to 8 week-old mice as known in the art. Acomposition comprising sex- and age-matched syngeneic apoptotic cellswas injected at 5×10⁶ cells per mouse and compared to syngeneic, sex-and age-matched mice that were injected with the vehicle (saline). Theroute of administration ws i.v., via the tail vein, without furthermanipulation. The cells were incubated at 37° C. in 5% CO₂ for 1 to 3hours to allow apoptotic changes to occur. After incubation, theapoptotic cells were injected into each mouse recipient. The injectionswere performed every week for a total of four to six injections.

Apoptotis. Apoptotsis of thymocytes or splenocytes was induced by eitherserum deprivation, 1 micromolar dexamethasone, or gama-irradiation (66rad). Apoptosis was confirmed by annexin-FITC staining by flowcytometry, DNA fragmentation and propidium iodide staining of fragmentedDNA.

Immune response. Serum samples were obtained immediately prior toimmunization and at two-weeks intervals following immunization. Theimmune response was evaluated by quantifying serum anti-ssDNA andanti-dsDNA by ELISA as known in the art. Sera were diluted 1:100 for theautoantibody screens.

Clinical and pathological evaluation. Mice were examined every day forclinical signs of disease and once a month for hematuria or proteinurea.After four months the mice were killed and the kidneys examinedhistologically and using fluorescent immune staining.

Results

Two groups of age- and sex-matched MRL/MpJ-Fas^(Ipr) mice were compared.In group 1, 200 microliter of saline containing syngeneic apoptoticcells were i.v. injected into each of one of five mice in a weeklyinterval for five times. In group 2, 200 microliter of saline (thevehicle for the first group) were injcted to the same number of mice atthe same time. IgG anti-ssDNA O.D. levels were conseutivly measured intwo weeks intervals and were comparable to the level beforeimmunization, mean O.D. of 0.096±0.018 in both groups (FIG. 1). Whencompared 10 weeks following the beginning of the immunization, miceimmunized with vehicle alone had, as expected from mice that developedlupus-like disease, higher levels, 0.308±0.029 (p<0.0000, studentt-test). However, mice injected with 1×10⁶ syngeneic apoptotic cells hadsignificantly reduced levels of autoantibodies, 0.193±0.017 (p<0.0000,student t-test). In FIG. 1: ? Group 1 (immunized with vehicle), 6weeks-old MRL/Ipr/Ipr ; 0 Group 2 (immunized with syngeneic apoptoticcells), 6 weeks-old MRL/Ipr/Ipr; {haeck over ( )} Group 1 (immunizedwith vehicle), at 16 weeks; ? Group 2 (immunized with syngeneicapoptotic cells), at 16 weeks.

In order to evaluate the increase in anti-ssDNA, serial bimonthly serasamples were evaluated simultaneously and showed for IgM, 0.198±0.017,0.205±0.02, and 0.300±0.033 for IgM; 0.378±0.037 for mice immunized withsaline; and 0.108(+0.03), 0.170(+0.07), 0.186(+0.04) and 0.203(+0.8) formice immunized with apoptotic cells. The O.D. statistical evaluationshowed that this decrease did not reach significance. In contrast, IgGanti-ssDNA levels became significantly decreased following theimmunization with syngeneic apoptotic cells: 0.132±0.09, 0.196±0.019,0.244±0.022, and 0.308±0.029 for mice immunized with saline, vs.0.109±0.012 (p=non-significant), 0.129±0.15, p<0.04), 0.166±0.014,(p<0.04), 0.192±0.17) (p<0.01), for mice immunized with syngeneicapoptotic thymocytes. As shown in FIG. 1, at age 16 weeks, a markeddecrease in anti-ssDNA was noted in all mice immunized with syngeneicapoptotic cells.

In order to see if autoantibodies even more specific for SLE weredecreased, anti-dsDNA was measured in all mice at the age of 6 weeks,before starting to immunize, and at 16-18 weeks of age, upon sacrifice.As shown in FIG. 2, anti-dsDNA was significantly reduced (p<0.00) inmice immunized with syngeneic apoptotic cells. Anti-dsDNA in average of0.599±0.026 measured in mice injected with saline and 0.358±0.038 inaverage in mice injected with syngeneic apoptotic cells. In FIG. 2: ?Group 1 (immunized with vehicle), 6 weeks-old MRL/Ipr/Ipr; 0 Group 2(immunized with syngeneic apoptotic cells), 6 weeks-old MRL/Ipr/Ipr;{haeck over ( )} Group 1 (immunized with vehicle), at 16 weeks; ? Group2 (immunized with syngeneic apoptotic cells), at 16 weeks.

To further compare if the clinical response follows the serological one,kidney-disease was compared in the two groups. None of the mice had anyevidence for proteinuria or hematuria as measured by urine-stick at 6weeks of age, before the immunization. At 16 weeks, mice immunized withsaline had significant elevations in proteinuria and hematuria asdemonstrated in Table 1. At 16 weeks all mice injected with saline alonedemonstrated glomerular disease manifested by proteinuria and hematuria.However, mice injected with syngeneic apoptotic cells showed markedimprovement (Table 1) consistent with the serological response. In twoout of five, no deterioration or very slight deterioration was noticed.In Table 1, Ipr-Apo=MRL/MpJ-Fas^(Ipr) mouse injected with syngeneicapoptotic cells; Ipr-S=MRL/MpJ-Fas^(Ipr) mouse injected with saline;and, C3H/SnJ is a normal mice used for control.

In order to confirm the clinical response, the extent of the diseaseprogression in the kidneys were evaluated by paraffin embedded andimmunofluorescent histological studies. Table 2 summarizes thehistopathological findings in blindly chosen three kidney sections ofeach group and demonstrates that mice injected with syngeneic apoptoticcells showed decreased involvement of disease in the glomeruli, vesselsand in the tubuli. In Table 2, Ipr-Apo=MRL/MpJ-Fas^(Ipr) mouse injectedwith syngeneic apoptotic cells; Ipr-S=MRL/MpJ-Fas^(Ipr) mouse injectedwith saline; and, C3H/SnJ is a normal mice used for control. TABLE 1Proteinuria and Hematuria in MRL/MpJ-Fas^(Ipr) Mice Injected withSyngeneic Apoptotic Cells Proteinuria Hematuria Mice 6 weeks 16 weeks 6weeks 16 weeks C3H/SnJ +1 +1 0 0 Ipr-S +1 +2 0 +2 Ipr-S +1 +3 0 +1 Ipr-S+1 +2 0 +3 Ipr-S +1 +2 0 +2 Ipr-S +1 +3 0 +1 Ipr-Apo +1 +2 0 +1 Ipr-Apo+1 +1 0 +1 Ipr-Apo +1 +1 0 +1 Ipr-Apo +1 +1 0 0 Ipr-Apo +1 +2 0 +1

TABLE 2 Histological and Indirect Immunofluoresence Evaluation for IgGDeposits in MRL/MpJ-Fas^(Ipr) Fluoresence Histology Indirect Tubuli GNVessels Tubuli GN C3H/SnJ — — — — — Ipr-S +2 +3 +1 +3 +3 Ipr-S +2 +2 +1− 2 +4 +3 Ipr-S +2 +2 − 3 +0 − 1 +3 +2 Ipr-S +1 +2 +0 +1 +1 Ipr-S +2 +1+0 +2 +1 Ipr-S +1 +1 +1 +3 +1

1. A method of treating a subject having an autoimmune disease,comprising the steps of: obtaining cells from the subject; inducing celldeath in said cells resulting in apoptotic and/or necrotic cells;administering to the subject an amount of said apoptotic and/or necroticcells effective to produce a modified immune response in said subject,thereby treating the subject with the autoimmune disease.
 2. A method oftreating a subject having an autoimmune disease, comprising the step ofadministering to the subject an amount of apoptotic and/or necroticcells effective to produce a modified immune response in said subject,thereby treating the subject with the autoimmune disease.
 3. The methodof claim 1 whereby the step of inducing cell death is obtained byexposing said cells to an apoptosis-inducing agent or to anecrosis-inducing agent.
 4. The method of claim 1 whereby the step ofinducing cell death is obtained by exposing said cells to anapoptosis-inducing treatment or to a necrosis-inducing treatment.
 5. Themethod according to any one of claims 1 to 4, whereby said modifiedimmune response is an increased tolerance to self-apoptotic cells. 6.The method according to claim 5, whereby said modified immune responsein said subject is a reduction in the tissue level of auto-antibodiesassociated with self-apoptotic cells.
 7. The method according to claim6, whereby said auto-antibodies are anti-nuclear antibodies, anti-singlestranded DNA antibodies, anti-double stranded DNA antibodies,anti-cardiolipin antibodies, anti-phosphatidylserine antibodies,anti-2GPI antibodies, anti-Sm antibodies, anti-RNP antibodies, oranti-Ku antibodies.
 8. The method according to claim 5, whereby saidmodified immune response in said subject is a reduction in the level ofinflammatory response.
 9. The method according to claim 8, whereby saidinflammatory response is associated with chemokines, cytokines,eicosanoids, complement proteins, C-reactive proteins, TNF, dendriticcells or a combination thereof.
 10. The method according to claim 1 or2, whereby said autoimmune disease is associated with an immune responseto self-antigens appearing on apoptotic cells.
 11. The method accordingto claim 10, whereby said autoimmune disease is systemic or discoidlupus, erythematosis, rheumatoid arthritis, polymyositis, or vasculitis.12. The method according to claim 1 or 2, whereby said cells arehematopoetic cells, thymocytes, splenocytes, lymphocytes, monocytes, acultured cell line or a combination thereof.
 13. The method according toclaim 3, whereby said apoptosis-inducing agent is a steroid, a peptide,a protein, a sugar, a lipid, an antibody, or a combination thereof. 14.The method according to claim 13, whereby said steroid is dexamethasone.15. The method according to claim 13, whereby said protein is perforin.16. The method according to claim 1 or 2, whereby said composition isadministered in combination with an immunosuppressing molecules.
 17. Apharmaceutical composition comprising an effective amount of apoptoticand/or necrotic cells, whereby the administration of said composition toa subject suffering from an autoimmine disease produces a modifiedimmune response in said subject.
 18. The composition according to claim17, wherein said modified immune response is a reduction in the tissuelevel of auto-antibodies associated with apoptotic cells in saidsubject.
 19. The composition according to claim 18, wherein saidauto-antibodies are anti-nuclear antibodies, anti-single stranded DNAantibodies, anti-double stranded DNA antibodies, anti-cardiolipinantibodies, anti-phosphatidylserine antibodies, anti-2GPI antibodies,anti-Sm antibodies, anti-RNP antibodies, anti-Ku antibodies, or acombination thereof.
 20. The composition according to claim 17, whereinsaid modified immune response is a reduction in the level ofinflammatory response.
 21. The composition according to claim 20,wherein said inflammatory response is associated with chemokines,cytokines, eicosanoids, complement proteins, C-reactive proteins, TNF,dendritic cells or a combination thereof.
 22. The composition accordingto claim 17, wherein said autoimmune disease is associated with animmune response to self-antigens appearing on apoptotic cells.
 23. Thecomposition according to claim 17, wherein said cells are fromautologous origin.
 24. The composition according to claim 23, whereinsaid cells are hematopoetic cells, thymocytes, splenocytes, lymphocytes,monocytes, or a combination thereof.
 25. The composition according toclaim 17, whereby said apoptotic and/or necrotic cells are obtained bycontacting the cells with an apoptosis-inducing agent or anecrotic-inducing agent or by exposing the cells to anapoptosis-inducing treatment or a necrosis-inducing treatment, or acombination thereof.
 26. The composition according to claim 25, whereinsaid apoptosis-inducing agent is a steroid, a peptide, a protein, asugar, a lipid, an antibody, or a combination thereof.
 27. Thecomposition according to claim 26, wherein said steroid isdexamethasone.
 28. The composition according to claim 26, wherein saidprotein is perforin.
 29. The composition according to claim 17, whereinsaid composition is suitable for administration via an intravenousroute, an intradermal route, a subdermal route, an intramuscular route,oral administration or a combination thereof.
 30. The compositionaccording to claim 17, wherein said composition is administered incombination with an immunosuppressing molecules.